The central hypothesis of Project I is that the spatial organization of signaling proteins is actively altered by mechanical forces and this provides a molecular scale mechanism for physical forces to directly regulate chemical signaling in cancer. The long-term goal of this proposal is tee develop a molecular level understanding of how mechanical forces can exert regulatory control over chemical signaling processes. We seek a fundamental understanding of hew the mechanical environment of a cell influences its intracellular chemical signaling. Te achieve this goal, we propose a highly multidisciplinary. hybrid physical and biological approach aimed at deconstructing how key chemical signal transduction pathways of significance in cancer can be responsive to mechanical inputs. The rationale for this is that the spatial organization of signaling proteins is altered in the different phases leading tee malignancy, and this is fundamentally net a chemical mutation in the structure of a protein, but rather a physical perturbation of protein organization on the macromolecular length scale (7, 2). The premise of our approach is that characterizing and controlling mechanical forces that drive receptor organization will allow us to elicit structural and functional phenotypes characteristic of defined phases of in cancer progression. We will target the EphA2 receptor signaling pathway as well as the Ras signaling module. These are chosen for their emerging roles in chemomechanical signal transduction. We will implement a combined approach that consists of 1) super-resolution imaging of hybrid cell-supported membrane junctions, 2) micro cantilever-based lateral force measurements of ligand-functionalized probes, and 3) nanoscissor laser surgery for cytoskeletal disruption. All three of these approaches will be employed in the context of the newly developed spatial mutation strategy, which provides unique opportunities tee mechanically perturb living cells with chemical specificity. Mathematical modeling is an essential part of all quantitative investigations and is integrated here as well.